Penetrability of proteins through the digestive system of Sarcophaga falculata blowfly.

On the background of the previous findings concerning the passage of a low molecular basic protein from cobra snake venom through the gut of a blowfly (Primor, N., Teitelbaum, Z. and Zlotkin, E. (1980) Biochim, Biophys, Acta 627, 71--81) several additional proteins were tested in the present study. With the aid of toxicity tests, radioiodinated proteins, gel filtration chromatography, and the usage of Sarcophaga falculata flies as test animals, the following information was obtained: 1. Out of the five species (representing four orders) of insects tested, only flies demonstrated the lethality by oral application of cobra snake venom. 2. The absence of oral toxicity of flies to phospholipase A2 (highly lethan by injection of cobra venom with molecular weight of about 13,000) was due to its impermeability through the digestive system. 3. About 2.2% of the orally applied low molecular weight basic neurotoxin (Mr 7000) from cobra venom crossed the gut and was found in the insects' hemolymph. 4. Bovine serum albumin (Mr 68,000) and an immunoglobulin (Mr 180,000), under the present experimental conditions, were found to be gut impermeable.

Functional membrane vesicles from the nervous system of insects. I. Sodium- and chloride-dependent gamma-aminobutyric acid transport.

(1) A synaptosomal fraction obtained from locust nervous tissue has been shown to possess an active gamma-aminobutyric acid transport mechanism. This activity is preserved and even enriched by the membrane vesicles derived from osmotically shocked synaptosomes. (2) Electron-microscopy examination indicates that the above membrane vesicles are derived predominantly from the neuronal plasma membrane and are devoid of any internal cellular organelles and components. Active transport of gamma-aminobutyric acid into these vesicles has been demonstrated with artificially imposed ion gradients as the sole energy source. (3) gamma-Aminobutyric acid transport can be driven by an Na+ gradient (out greater than in) and/or by a gradient of Cl- (out greater than in). This process is absolutely dependent on the simultaneous presence of both types of ion in the external medium. The stimulation of the process by valinomycin indicates that gamma-aminobutyric acid transport is an electrogenic process which is stimulated by a membrane potential (interior negative).

Penetrability of orally toxic protein from cobra venom through the gut of a blowfly.

The oral toxicity of a radioiodinated toxic polypeptide isolated from a cobra snake venom as assayed by Sarcophaga falculata blowflies coupled with assays on competitive displacement have indicated that: (a) During 3--4 h 8% of the orally active toxin is able to pass through the digestive system of the fly; (b) the orally active toxin after passing the gut binds to body tissues. The strong affinity of the toxin to tissue membranes explains its absence in the insect's hemolymph following oral applications as well as injection. The removal of traces of phospholipase A, which is extremely toxic, by injection of the orally active toxin has significantly lowered its injection toxicity without affecting its oral toxicity, thus indicating the absence of any interaction with phospholipases in oral toxicity. This conclusion was supported by additional experimentation.

Sodium channel polypeptides in central nervous systems of various insects identified with site directed antibodies.

Immunoprecipitation, radiophosphorylation and SDS-PAGE autoradiography enable the characterization of sodium channel polypeptides in the central nervous system of insects belonging to four phylogenetically distinct orders: grasshoppers, cockroaches, flies and moth larvae. It has been shown that the insect sodium channels: (1) Are recognized by the previously described (Gordon et al. (1988) Biochemistry 27, 7032-7038) site directed antibodies corresponding to a highly conserved segment linking the homologous domains III and IV in the vertebrate sodium channel alpha subunits. (2) Serve as substrates for phosphorylation by cAMP-dependent protein kinase. (3) Are devoid of disulfide linkage to smaller subunits unlike sodium channels in vertebrate brain. (4) Are glycoproteins as shown in the grasshopper by the decrease of apparent molecular weight following endoglycosidase F treatment and specific binding to the lectins concanavalin A and wheat germ agglutinin. (5) Reveal a diversity with regard to their (a) apparent molecular masses which range from 240 to 280 kDa and (b) V8 proteinase digestion phosphopeptides indicating either differences in the positioning of the enzymatic cleavage and/or phosphorylation sites. These results provide the first evidence for structural diversity of sodium channel subtypes among various insect orders and are compared to their mammalian counterparts.

Liposomes as a model for the study of the mechanism of fish toxicity of sodium dodecyl sulfate in sea water.

The mechanism underlying the shark repellency of SDS was studied by comparing it with the shark nonrepelling detergent, Triton X-100. The findings can be summarized as follows: (1) The effective concentration of SDS for termination of shark tonic immobility (an immediate and fast response) was close to its critical micellar concentration in sea water (70 microM). The fish lethal concentrations (LD50) were far below the CMC value for SDS, and at CMC level for Triton X-100. (2) In sea water SDS possesses a strong affinity for lipid membranes, expressed in a lipid sea water partition coefficient (Kp) of about 3000. (3) In liposomal systems examined by assays of turbidity, fluorescence resonance energy transfer and kinetics of carboxyfluorescein (CF) release, the pattern of SDS induced changes in the phospholipid bilayer suggests: (a) absence of vesicle-vesicle fusion; (b) occurrence of vesicle size increase, and (c) nonlytic gradual release of CF above and below its CMC values. In contrast, Triton X-100 above its CMC induces membrane solubilization. (4) Assays coupling CF release from liposomes to potassium diffusion potential induced by valinomycin indicate that SDS related CF release can also be attributed to a specific mechanism such as cation pore formation and not only to membrane solubilization. The hypothesis of pore formation by SDS is discussed.

Binding of an alpha scorpion toxin to insect sodium channels is not dependent on membrane potential.

The insect-specific Lqh alpha IT toxin resembles alpha scorpion toxins affecting mammals by its amino acid sequence and effects on sodium conductance. The present study reveals that Lqh alpha IT does not bind to rat brain membranes and possesses in locust neuronal membranes a single class of high affinity (Kd = 1.06 +/- 0.15 nM) and low capacity (Bmax = 0.7 +/- 0.19 pmol/mg protein) binding sites. The latter are: (1) distinct from binding sites of other sodium channel neurotoxins; (2) inhibited by sea anemone toxin II; (3) cooperatively interacting with veratridine; (4) not dependent on membrane potential, in contrast to the binding sites of alpha toxins in vertebrate systems. These data suggest the occurrence of (a) conformational-structural differences between insect and mammal sodium channels and (b) the animal group specificity and pharmacological importance of the alpha scorpion toxins.

Characterization of the transcript for a depressant insect selective neurotoxin gene with an isolated cDNA clone from the scorpion Buthotus judaicus.

The poly(A)+ mRNA isolated from the venomous terminal segments of the scorpion Buthotus judaicus was reversed transcribed into cDNA. PCR amplification of the cDNA in presence of oligonucleotide primers prepared on basis of the known amino acid sequence of the depressant insect toxin II yielded a 125 bp long product. This fragment was cloned and its sequence determined. The deduced amino acid sequence has revealed a complete homology with the amino acid sequence of the toxin. This clone was used to probe a Northern blot resolving the poly(A)+ and poly(A)- fractions derived from the scorpion. An organ specific 360 nucleotide transcript which might be the processed product of a approximately 4.0 kb precursor was elucidated. This cDNA clone may pave the way for a molecular genetic approach to study the structure-function relationship of scorpion selective insect toxins.

Functional expression of an alpha anti-insect scorpion neurotoxin in insect cells and lepidopterous larvae.

The Leiurus quinquestriatus hebraeus alpha anti-insect toxin (Lqh alpha IT) cDNA was engineered into the Autographa californica Nuclear Polyhedrosis Virus (AcNPV) genome. Insect cells infected with the recombinant virus secreted a functional Lqh alpha IT polypeptide. Spodoptera littoralis and Heliothis armigera larvae injected with recombinant budded virus, showed typical intoxication symptoms. This recombinant virus showed enhanced insecticidal potency against H. armigera larvae compared with wild type AcNPV. The present expression system will facilitate: (1) the future elucidation of structural elements involved in its prominent anti-insect toxicity; and (2) the future design of genetically modified alpha toxins with improved anti-insect selectivity.

AaIT: from neurotoxin to insecticide.

AaIT is a single chain neurotoxic polypeptide derived from the venom of the Buthid scorpion Androctonus australis Hector, composed of 70 amino acids cross-linked by four disulfide bridges. Its strict selectivity for insects has been documented by toxicity, electrophysiological and ligand receptor binding assays. These last have shown that various insect neuronal membranes possess a single class of non-interacting AaIT binding sites of high affinity (K(D) = 1-3(n)M) and low capacity (0.5-2.0 pmol/mg prot.). The fast excitatory paralysis induced by AaIT is a result of a presynaptic effect, namely the induction of a repetitive firing in the terminal branches of the insect's motor nerves resulting in a massive and uncoordinated stimulation of the respective skeletal muscles. The neuronal repetitive activity is attributed to an exclusive and specific perturbation of sodium conductance as a consequence of toxin binding to external loops of the insect voltage-dependent sodium channel and modification of its gating mechanism. From a strictly agrotechnical point of view AaIT involvement in plant protection has taken the following two complementary forms: firstly, as a factor for the genetic engineering of insect infective baculoviruses resulting in potent and selective bio-insecticides. The efficacy of the AaIT-expressing, recombinant baculovirus is attributed mainly to its ability to continuously provide and translocate the gene of the expressed toxin to the insect central nervous system; secondly, based on the pharmacological flexibility of the voltage-gated sodium channel, as a device for insecticide resistance management. Channel mutations conferring resistance to a given class of insecticidal agents (such as the KDR phenomenon) may greatly increase susceptibility to the AaIT expressing bioinsecticides. Thus the AaIT is a pharmacological tool for the study of insect neuronal excitability and chemical ecology and the development of new approaches to insect control.

The pharmacological flexibility of the insect voltage gated sodium channel: toxicity of AaIT to knockdown resistant (kdr) flies.

AaIT is an insect selective neurotoxic polypeptide shown to affect insect neuronal sodium conductance by binding to excitable sodium channels. In the present study the paralytic potency of AaIT to wild type and various mutant strains of houseflies (Musca domestica) and fruitflies (Drosophila melanogaster) was examined and it has been shown that: On the basis of body weight when compared to published data on Sarcophaga falculata blowflies, the Musca and Drosophila flies reveal at least two orders of magnitude decreased susceptibility to the AaIT. When compared to wild type flies the toxicity of AaIT is greatly altered in knockdown resistant fly strains which are mutated in their para gene encoding the voltage gated sodium channel. Several strains, with genetically mapped para mutations conferring pyrethroid resistance, exhibited opposing response to AaIT. The para ts2 Drosophila strain, with a point of mutation in domain I of the para gene conferring a 6-fold resistance to deltamethrin also showed about 15-fold tolerance to AaIT. On the other hand the Musca kdr and super-kdr flies, with a single or a double point mutation, respectively in domain II of the para gene, are about 9- and 14-fold more susceptible to AaIT, respectively. The above data are interpreted in terms of the pharmacological diversity and flexibility ("allosteric coupling") of voltage gated sodium channels and their implications for the management of pesticide resistance are discussed.

Insect tolerance to a neurotoxic polypeptide: pharmacokinetic and pharmacodynamic aspects

Androctonus australis insect toxin (AaIT) is an insect-selective neurotoxic polypeptide from scorpion venom used to probe insect Na+ channels and to design insecticidal recombinant baculoviruses. When injected into susceptible insects (such as flies or cockroaches), nanogram doses of the toxin induce a rapid paralysis within seconds. More tolerant insects respond to microgram doses by developing either a slow progressive paralysis, as in lepidopterous larvae, or a rapid but reversible paralysis, as in Trachyderma philistina, a tenebrionid beetle. Using toxicity and binding assays, microscopy and chromatography, we show that the tolerance of insects to AaIT occurs at both the pharmacokinetic and pharmacodynamic levels. Pharmacokinetic effects occur in Trachyderma philistina in which the toxin undergoes a progressive process of degradation and elimination from the hemolymph, resulting in the loss of 95­97 % of toxin activity 6 h after injection. The pharmacodynamic aspect was demonstrated in studies of the kinetics of binding dissociation of [125I]AaIT from neuronal membranes of susceptible and tolerant insects. Stable binding is shown in susceptible insects such as cockroaches and locusts, which have a dissociation half-time of approximately 9 and 5 min, respectively. This contrasts strongly with the fast half-time of dissociation of 7 s for Spodoptera littoralis larvae and 9 s for Trachyderma philistina, which are both relatively tolerant to AaIT. These differences in binding kinetics may reflect a structural and functional diversity of Na+ channels in different insects that is responsible for their diverse susceptibility to neurotoxic polypeptides.

Solubilization and characterization of the insect neuronal sodium channel.

Locust neuronal sodium channels were solubilized by 1% cholate and 0.2% Triton X-100, and their functionality was monitored by [3H]saxitoxin (STX) binding assays. About 40% of STX binding activity was recovered in the solubilized fraction without affecting affinity (Kd = 0.5 nM) and the time and temperature dependent STX binding activity was significantly stabilized in the presence of 20 nM STX. Partial purification by an anion exchange resin yielded a 20% recovery and a 3.5 times increase in the specific STX binding activity. Identification of the locust solubilized sodium channels by immunoprecipitation and radiophosphorylation revealed a Mr of 245,000 on SDS-PAGE. The present solubilized preparation will enable the study of the unique pharmacology of insect sodium channels.

A new bioassay reveals mollusc-specific toxicity in molluscivorous Conus venoms.

Contraction of the foot pedal of a limpet snail is described as a new and quantifiable bioassay for mollusc paralysis. This bioassay was used for screening the venoms of seven different species of Conus snails. Comparison of the results of the limpet assay with those obtained from fish and blowflies shows a correlation between the feeding specificities and venom toxicities of these Conidae. The limpet bioassay should be useful for identification and monitoring of the purification of new toxins active on molluscan systems.

The cDNA sequence of a depressant insect selective neurotoxin from the scorpion Buthotus judaicus.

A 400 nucleotide cDNA clone encoding the depressant insect toxin of the scorpion Buthotus judaicus (BjIT2), was isolated. DNA sequence analysis suggests that the toxin is a processed product of a precursor composed of: (1) a 21 amino acid residue signal peptide; (2) a 61 amino acid region of the mature toxin; and (3) an additional Arg-Lys-Lys tail at the carboxy terminus prior to a termination codon. Comparison between the precursor polypeptides of BjIT2 and another depressant insect toxin derived from the scorpion Leiurus quinquestriatus hebraeus (LqhIT2) shows similarities in their hydropathic profiles.

Nucleotide sequence and structure analysis of a cDNA encoding an alpha insect toxin from the scorpion Leiurus quinquestriatus hebraeus.

A approximately 370 base pair cDNA encoding the alpha insect toxin Lqh alpha IT of the scorpion Leiurus quinquestriatus hebraeus was cloned and sequenced. The deduced amino acid sequence for the putative mature polypeptide is identical to the protein sequence determined chemically (Eitan et al., Biochemistry 29, 5941, 1990). A 19 amino acid signal peptide precedes the 64 amino acid long toxin. Two additional amino acid residues that do not correspond to the purified toxin are found at the COOH-terminus and may imply post-translational modification. The signal peptide region in the present clone differs obviously from that encoding the depressant insect toxin LqhIT2 derived from the same venom, but strongly resembles the leader peptide sequence of an alpha-mammal toxin from the scorpion Androctonus australis.

Positive cooperativity among insecticidal scorpion neurotoxins.

The insecticidal activity of scorpion neurotoxic polypeptides increased 5-10-fold with no apparent increase in mammalian toxicity when a combination of two toxins was injected. Synergistic combinations could be predicted from binding studies and competitive displacement assays. Our results indicate that simultaneous expression in baculovirus or other transgenic organisms of the synergistic combinations of insecticidal toxins may result in more potent insect-selective biopesticides.

Effect of Leiurus quinquestriatus hebreus venom on calcium and deoxyglucose uptake in cultured cardiac cells.

The effects of scorpion venom Leiurus quinquestriatus hebreus were studied on cardiac cells grown in culture. The venom (30 micrograms/ml) increased significantly (P < 0.05) Ca2+ uptake into intact cardiocytes and to sarcoplasmic reticulum of skinned cells. [3H]Deoxyglucose uptake was also increased significantly (P < 0.05) in venom treated cardiocytes. It was found that fractions I and III of the venom, separated by gel filtration and ion exchange chromatography, are responsible for the increased Ca2+ uptake by the sarcoplasmic reticulum, whereas fraction IIb, III and IV are responsible for the accelerated rate of uptake of 45Ca and [3H]deoxyglucose by intact cells. Ca channel blockers prevented these effects and similar results were obtained by propranolol. Thus, it is concluded that the venom exerts its effect through activation of beta-adrenoceptors which causes the opening of L-type Ca channels.

Gene construction, expression and functional testing of an inotropic peptide from the venom of the black scorpion Hottentotta judaicus.

Anti-insect depressant toxins represent a subfamily of scorpion venom-derived ß-toxins that are polypeptides composed of 61-65 amino acid residues stabilized by four disulfide bridges. These toxins affect the activation of voltage-sensitive sodium channels (NaScTx) and exhibit the preferential ability to induce flaccid paralysis in insect larvae. Here we demonstrate the recombinant expression of the novel cardiac inotropic peptide (Bj-IP) that was classified as an anti-insect depressant ßNaScTx isolated from the venom of Hottentotta judaicus. By using "splicing by overlap extension" (SOE)-PCR, allowing for the first time one step de novo synthesis of long-chain scorpion toxin genes, we generated a codon-optimized DNA fragment of Bj-IP for cloning into the Escherichia coli vector pQE30. Moreover, the gene of interest was fused to a 6xHis coding DNA sequence. Subsequent recombinant expression was performed in E. coli KRX. The purification of the polypeptide was achieved by a combination of NiNTA agarose columns and RP (C(18)) high-performance liquid chromatography. The purified fusion protein was digested with factor Xa resulting in the elution of Bj-IP. The yield of recombinant Bj-IP expression was approximately 4.5 mg per liter of culture. Mass spectrometry confirmed the theoretical total mass of Bj-IP (6608 Da). Tag-free Bj-IP was refolded in guanidine chloride buffer with a glutathione redox system which was supplemented with different additives at 16 °C. Supplementation with 10% glycerol produced Bj-IP folding forms that exhibited reproducible biological activity in mouse cardiomyocytes. Cell contractility was increased by almost 3-fold and decay kinetics were hasten by 47% after administration of Bj-IP. Taken together, here we show the recombinant expression of the functionally active cardiac inotropic peptide Bj-IP, a new ßNaScTx from H. judaicus, for promising pharmacological applications. Furthermore, our data suggest that the use of SOE-PCR may help to facilitate in future the high throughput of cloning and/or modification of scorpion toxin genes.